The origin(s), fate, and physiologic actions of endogenous acetylcholine (ACh) in the aqueous humor are little understood but may be important, particularly since cholinergic drugs are used to reduce intraocular pressure in glaucoma. Specifically, the physiological role of ACh with regard to the regulation of intraocular pressure may be more significant than heretofore appreciated, particularly since the endogenous ACh concentration in the aqueous humor is of the same order of magnitude as the average therapeutic pilocarpine concentration achieved in the anterior chamber with topical application. Preliminary investigations have shown that it is possible to reliably measure the acetylcholine (ACh) concentration in anterior chamber fluid aqueous humor by paracentesis obtained from single eyes of rabbits and cats. ACh is normally present in a number of ocular tissues. Using paired comparison methodology, the anterior chamber fluid ACh concentration in rabbits, cats, and monkeys will be measured with regard to: (1) time of day (diurnal variation); (2) intraocular pressure; (3) effects of pre-and post-ganglionic oculomotor (cranial nerve III) neurotomies; (4) effects of topically applied drugs used for glaucoma therapy; (5) effect of topically applied dexamethasone (which induces elevation of intraocular pressure in humans), and (6) effects of primary paracentesis on a subsequent paracentesis. The comparison of the three species and information concerning the safety of the paracentesis method will be the basis for a subsequent proposal to extend the assay to specimens obtained from human eyes. Other subsequent proposals will seek to expand the ability of this laboratory to measure ACh and Acetylcholinesterases (AChE) in other ocular tissues.